ES2306811T3 - ANTI-INFECTIVE CARBOHYDRATES. - Google Patents

Abstract

Neutral, linear or branched oligosaccharides (I), having a structure based on galactose, glucose and N-acetylated galactosamine and/or glucosamine moieties, are used for reducing or inhibiting the invasion and infection of mammalian cells by pathogens and for combating diseases caused by the pathogens. The use of neutral, linear or branched oligosaccharides (I) is claimed for reducing or inhibiting the invasion and infection of mammalian cells by pathogens and for combating diseases caused by the pathogens, where (I) comprises: (a) a base unit of formula R-(Gal(R)-HexNAc)-Gal(R)-Glc; (b) 0-19 further units of formula R-(Gal(R)-HexNAc)-, directly or indirectly linked to the base units; and (c) a terminal (Gal-HexNAc)-unit. Gal : galactose unit Glc : glucose unit; HexNAc : N-acetylated galactosamine (GalNAc) or N-acetylated glucosamine (GlcNAc) unit; R : deoxyhexose residue, beta 1-3 or beta 1-6 glycosidically bonded to the HexNAc moiety of the adjacent (Gal-HexNAc) unit (b) or to the galactose moiety of a terminal (Gal-HexNAc) unit (c); or is absent; a terminal unit (c), to which at least one deoxyhexose unit R is bonded, may carry further deoxyhexose residues. Independent claims are also included for the following: (i) nutritional, dietetic or pharmaceutical compositions (other than human milk), containing (I), conventional additives (such as diluents, humectants, thickeners, flavorings, sweeteners and/or carriers) and optionally other active agents; and (ii) a corresponding treatment method, involving administration of (I) or compositions as in (i) to humans or animals. ACTIVITY : Antibacterial; Virucide. MECHANISM OF ACTION : Blocking or modifying cell surface receptors; inhibiting adhesions of pathogens to cell surfaces.

Description

Anti-infective carbohydrates

The present invention relates to the use of straight or branched neutral chain oligosaccharides, to prevent the invasion and infection of mammalian cells by pathogens and to combat diseases caused by such pathogens, and to media food, dietary and pharmaceutical containing these oligosaccharides

The adhesion of pathogenic organisms as well as of substances harmful to cells on the surfaces of mammalian cells is the first step and an indispensable condition for an infection or cell damage. The interaction between pathogens and cells takes place through a relationship ligand-receptor. Glycostructures play a important role in these relationships or exchange effects.

A possibility to influence such relationships ligand-receptor lies in blockage or change structural of the corresponding receptors, on the surface of the cells or on the ligands.

In specific test systems, different Carb mixtures have proven very effective in reducing e even to prevent the adhesion, for example, of microorganisms to the cell surface: Kunz, C .; Rudloff, S. Acta Paediatr. 1993, 82, 903-912. Other substances, as per example Lewis structures as carbohydrate ligands of selectin (adhesion protein on the endothelium and lymphocytes),  modulate the interaction of lymphocytes with the endothelium, by example, in the framework of Rolling, Homing and invasion in processes of initiation (Norman, K.E .; Anderson, G.P .; Kolb, H.C .; Ley, K .; Ernst, B. Blood 1998, 91, 475-483). The lectin that recognizes galactose, galectin, plays an important role additional physiological in relation to cellular functions fundamental as well as with specific functions such as membership, migration, chemotaxis, proliferation, apoptosis, growth of neurites (Cooper DN & Barondes SH, Glycobiology 1999 9 (10) 979-984). For the Nematoden C. elegans you can indicate that your LEC-1 galectin can bind with different specificity different oligosaccharide derivatives that They contain galactose. (Arata Y. Hirabayashi J. Kasai K, JBC, 2001: 276, 5, 3068-3077). By employing Galactose-specific lectins in a mouse model could reduce the lethality of an experimental listeriosis (Stoffel B, Beuth J, Pulverer G, Zentralbl. Bakteriol 1996, 284: 439-442). The adherence of microorganisms on host cells can also be the cascade trigger of signal in pathogens foreign to the body as well as in cells own body.

There is another possibility to reach a influence of cellular processes at the level of molecular biology. It can lead to it being reduced or prevented, for example, from being disconnect defense mechanisms in mammalian cells or the expression of virulence mechanisms in microorganisms pathogens (eg disconnection of virulence genes in bacteria by blocking the central regulators). From this way, using certain carbohydrates as cellobiose you can successfully stop the expression of certain surface structures of pathogenic listerias, which are responsible for host cell invasion (Park SF Kroll RH Mol Microbiol 1993 8: 653: 661; WO- A 94/02586).

It is an objective of the present invention to show how can with the help of oligosaccharides be reduced or prevented invasion and infection of mammalian cells with listerias and how can fight effectively against diseases caused by such listerias.

This objective is met by writing in The claims.

According to the invention, they are conducted for special oligosaccharide application to achieve the task of invention. In the following, these oligosaccharides are described as oligosaccharides according to the invention.

According to the invention, they can be used single oligosaccharides alone, in accordance with the invention, or several oligosaccharides according to the present invention in admixture. In addition, it is possible to use an oligosaccharide according to the invention or several oligosaccharides according to the invention or also a mixture of numerous oligosaccharides according to the invention together with other carbohydrates that are not counted among oligosaccharides in accordance with the invention, in the form of a mixture of carbohydrates

The oligosaccharides according to the invention They exhibit a base unit of the following general formula:

one

in it Gal represents a unity of monosaccharide galactose, Glc a unit of glucose monosaccharide and HexNAc monosaccharide unit galactosamine or glucosamine N-acetylated (may also be abbreviated as GalNAc or GlcNAc).

The radical R in the general formula I has no to be necessarily present with which, within the framework of This document, a hydrogen atom should not be taken as a radical. Therefore, simple oligosaccharides consistent with the Invention have the following general formula:

[Gal - HexNAc] --- Gal-Glc.

Among these oligosaccharides is the lacto-N-tetraose (Gal? 1-3GlcNAc? 1-3Gal? 1-4Glc, LNT) preferred according to the invention.

In these last oligosaccharides the unit [Gal - HexNAc] represents a terminal unit. This can be linked in the monosaccharide galactose unit (Gal) with a deoxyhexose (preferably α1-2 glycosidic) or with two deoxyhexose. The radical R or the radicals R in the general formula I can / can in this case therefore be one of such radicals deoxyhexose, which or which are a rhamnose radical and fucose

Finally, the compounds include the lacto-N-tetraose preferred from according to the invention, which is derivatized by fucose in a glycosidic α1-2 bond in the unit monosaccharide terminal galactose unit [Gal-HexNAc]; in it is lacto-N-fucopentaose I (LNFP I).

The radicals R of the basic unit of the general formula I can also represent junction points of the general formula II to other units [Gal-HexNAc]:

2

The radicals R of this unit [Gal - HexNAc] of general formula II linked directly to the basic unit of general formula I (one or two radicals R may be present per unit) can again mean points of attachment to others units [Gal - HexNAc] of the general formula II, which then they are indirectly linked to the basic unit of the general formula I. Also, that other units [Gal - HexNAc] of the general formula II can then again have junction points, etc. The units [Gal - HexNAc] are linked to a link glycosidic? 1-3 or? 1-6. Among these oligosaccharides are counted by example those of the following general formula, to name just Some:

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3

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4

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5

       \ vskip1.000000 \ baselineskip
    

6

Y

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7

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In these formulas the radicals R possess the meanings given above. Thus, for example, not all R radicals be present. In addition, one or both radicals R or also all radicals R can represent a union with the next unit [Gal - HexNAc], which again in each case it can show a radical R or two radicals R or also not have none of such radicals R. In short, in the corresponding oligosaccharides  with the invention up to 20 units of [Gal - HexNAc] and therefore 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 of such units, where the first of such units belong to the basic unit of the general formula I and the other units until 19 correspond to the general formula II. The oligosaccharides according to the invention can, therefore therefore, represent straight or branched chain molecules.

If for demonstration purposes we accept by once in the formulas cited above (III) to (VII) none of the radicals R represents a link to the neighboring units [Gal - HexNAc], then the compounds of the formula (III) and (IV) in each case they only have a terminal [Gal - HexNAc] terminal, that is the observed more to the left, while the other units are intermediate. The compounds of the formula (V), (VI) and (VII) have in each case two of such terminal units, where the compound (V) also shows two and compound (VII) shows a unit intermediate of general formula II. In these terminal units R radicals can also represent a deoxyhexose. For the either, 1, 2, 3 (as long as they are present) or all terminal units [Gal - HexNAc] of oligosaccharides according to the invention may have one such deoxyhexose radical or Two such radicals.

In addition, one or more (as long as they are present) terminal unit (s) of [Gal-HexNAc], in which or in which the or radical (s) R represents a radical of deoxyhexose, may have in the monosaccharide unit of HexNAc another deoxyhexose radical.

Among the preferred oligosaccharides according with the invention they are such that they satisfy one, two, three or four of the following criteria:

-

He deoxyhexose radical, as long as it is present, of the unit of galactose monosaccharide of the unit (s) terminal (s) [Gal-HexNAc] is linked to she with a glycosidic α1-2 bond,

-

at deoxyhexose radical, as long as the unit of monosaccharide galactose of the unit (s) terminal (s) [Gal-HexNAc] is a radical rusty and fuzzy.

-

he another deoxyhexose radical, as long as it is present, of the unit of monosaccharide HexNAc of the unit (s) terminal (s) [Gal-HexNAc] is linked to it through an α 1-3 or α link 1-4 glycosidic

-

at another deoxyhexose radical, as long as it is present, the unit of HexNAc monosaccharide of the unit (s) terminal (s) [Gal-HexNAc] is a radical rusty and fuzzy.

The oligosaccharides according to the invention are common and known compounds. Thus, among the oligosaccharides used in accordance with the invention are LNT and LNFP I the which are present in human milk.

Surprisingly it was found that oligosaccharides according to the invention at least decrease or they even prevent the invasion and infection by listerias of the mammalian cells and can be used to fight against the diseases caused by such listerias.

It was found, for example, that oligosaccharides according to the invention can prevent the invasion and infection of mammalian cells by listeria, in particular Listeria monocytogenes . The results of the investigation clearly show that neither the course of phagocytosis per se, nor the replication of the listerias included are inhibited. Among others, the oligosaccharide according to the invention LNFP I has been tested as a particularly strong inhibitor.

Therefore, the LNFP I pentasaccharide showed a strong, although dose dependent, inhibitory effect against listerias. Compounds that are structural isomers of LNFP I, to which belong for example the oligosaccharides LNFP II and LNFP III as well as LNFP V, do not show any such effect against it. The oligosaccharides LNFP II and LNFP III are also found as the LNFP I in human milk.

The oligosaccharides according to the invention they can be used not only as free oligosaccharides (ie with a reducing end), but also immobilized or adsorbed on a support. These supports can be a peptide / protein (for example BSA), a lipid, (glycolipid, ceramide), a polymer or a biopolymer (e.g. carbohydrate dendrimer, polysaccharide polyacrylamide) or any other aglycone.

The oligosaccharides according to the invention, it is only a free oligosaccharide or an oligosaccharide bound on a support, can be incorporated into different foods, media Dietary and pharmaceutical media. All these means can be present in liquid or solid form. The concept used here of food product covers not only real foods but also supplements, drinks as well as foods that include Baby food and infant formulas. The food concept for babies or infant formulas covers all foods artificially produced, although not human milk. It is understood by "artificial" foods that are produced from materials Vegetable raw and animal origin. These foods can be administered in any way to a person or an animal. Between these are also counted administration as feed by tube in the stomach.

The oligosaccharides according to the invention they can for example be added as additives to the following products, where this list is not closed: milk and products dairy, baby food and infant formulas, bars chocolate, yogurt drinks, cheese products, sausages and meat, structural foods, parenteral foods and products for pregnant women

The oligosaccharides according to the invention they can also be administered in the form of a pharmaceutical medium alone or together with several active substance (s) additional. For example, if they were formulated as tablets / envelopes For the formulation of such pharmacists can used adjuvants, supports, diluents, humectants, thickeners, flavorings, sweeteners, etc.

The pharmaceutical media can be administered in all common ways to a patient (i.e. human and animal). However, properly they are means that are formulated for oral, lingual, nasal administration, intestinal, bronchial, vaginal, topical (skin and mucosa) and per os (via oral) appropriate and corresponding to the type of administration.

Food, dietary means and means Pharmacists containing at least one chord oligosaccharide with the invention they can be used, among others, for prevention and treatment of infections of the gastrointestinal tract, by example in listeriosis, blood system, pathways respiratory, urogenital tract as well as cavity nasopharyngeal and for the care of the endothelium, epithelium and mucous membranes. Therefore they can find use applied topically on the skin or also on the mucous membranes. Among these mucous are nasal, intestinal, bronchial and vaginal count. So, the oligosaccharides according to the invention can be for example incorporated into a mouthwash. As target groups for oligosaccharide according to the invention can be mentioned all the stages of life, from newborns to older adults. The care and treatment of pregnant, sick, people Weak and elderly, they are particular demands of use, in the which is especially important to avoid for example a listeriosis

The oligosaccharides according to the invention and for example LNFP I are produced according to chemical methods or suitable and known enzymes, or according to a coupling of both technologies. This occurs systematically from the monosaccharide components or by modification of raw materials suitable oligosaccharide. In enzymatic synthesis both are used transferases (Leloir or not Leloir) as well as hydrolases (reverse hydrolyses or transglycosylation). Therefore the enzymes can be free or also incorporated (for example membrane reactor) or be covalently bonded on a support (for example spheres, chromatographic material, or membranes of filtration). It is also possible to use pro cells for synthesis or eukaryotic, as long as these cells have enzymes adequate. In relation to other individual units for the production of oligosaccharides according to the invention, by example refer to Carbohydrates in Chemistry and Biology (Herausgeber Ernst, Hart, Sinay) Wiley VCH-Weinheim 2000 Vol. I-IV).

Listed below are copies of Dietetics and pharmaceuticals containing the corresponding oligosaccharides with the invention

Example 1

For the production of envelopes they are mixed in each dry case 100 mg of LNFP I with 990 mg of maltodextrin and then They are packed in envelopes. These envelopes are administered three times daily at lunchtime.

Example 2

It is mixed by the one known per se production, a known complete food (i.e. Milupa® HN 25, balanced diet) in the form of a pearl that has 18.8 g of protein, 8.6 g of fat, 62.8 g of carbohydrates, 3.3 g of minerals and vitamins with LNFP I in an amount such that 50 mg of LNFP I are present in 100 g of the ready pearl.

For the production of a complete food liquid, 100 ml of the known liquid complete food are mixed Milupa HN 25 (2.3 g of protein, 1.6 g of fat, 8.5 g of carbohydrates, 37 g of minerals, vitamins) with 7 mg of LNFP I.

Example 3 Product for pregnant women

It occurs in the form known per se a effervescent tablet (final weight 4.15 g) (Neovin® by Milupa) by adding 200 to 500 mg of LNFP I. It dissolves daily. One tablet in water and you drink.

Example 4 Product for elderly and weak people

It is produced in the manner known per se a powder-balanced structural food (Dilsana® from Milupa) which has 22.5 g of protein, 7.7 g of fat, 60.8 g of carbohydrates, 5.4 g of minerals and vitamins by incorporation of 100 mg to 1000 mg of LNFP I per 100 g of powder. Daily dissolve in 150 ml of water up to 3 x 50 g of Food and is administered.

Example 5 Tea

100 g of an instant tea powder are mixed Commonly produced with 2 g of LNFP I. Dissolve 3.8 g of powdered tea in 100 ml of hot water and administered three times per day.

Example 6

A dairy food is manufactured in the common way for infants adapted in protein (Aptamil® from Milupa) that has 11.8 g of protein, 56.9 g of carbohydrates, 24.9 g of fat, 2.5 g of minerals, vitamins and 45 mg of taurine in the form of a pearl, which is mixed with 100 mg to 1000 mg of LNFP I per 100 g of Dairy foods for infants.

Claims (13)

1. Use of neutral chain oligosaccharides straight or branched, for the production of a food, medium dietary and pharmaceutical means, to reduce or prevent the invasion and infection of mammalian cells by listeria and to fight against diseases caused by listeria, oligosaccharides that are composed of a basic unit of the following general formula I: 8 and 0 to 19 additional units linked directly or indirectly with it, of the following formula general II 9 Where Gal represents a monosaccharide unit galactose, Glc represents a monosaccharide unit glucose, HexNAc represents a monosaccharide unit galactosamine or N-acetylated glucosamine (GalNAc or well GlcNAc), the radicals R in each case independently they represent, or are not present, a glycosidic bond β1-3 or β 1-6 to the unit of monosaccharide HexNAc of the following unit [Gal-HexNAc] of the general formula II, and in a terminal unit [Gal-HexNAc] a radical of deoxyhexose bound to the monosaccharide galactose unit of this terminal unit [Gal-HexNAc], and a terminal unit [Gal-HexNAc], in which the radical (s) R represents (n) a deoxyhexose radical, in which the HexNAc monosaccharide unit may have another radical deoxyhexose 2. Use according to claim 1, characterized in that the oligosaccharides correspond to the following general formula 10 where the radicals R possess the given meanings above. 3. Use according to claims 1 or 2, characterized in that the oligosaccharides satisfy at least one of the following criteria:

-

he deoxyhexose radical, as long as it is present, of the unit of terminal unit galactose monosaccharide [Gal-HexNAc] is linked to her in a way α1-2 glycosidic,

-

at deoxyhexose radical, as long as the unit of terminal unit galactose monosaccharide [Gal-HexNAc] is a rhamnose and fucose radical

-

he another deoxyhexose radical, as long as it is present, of the unit of HexNAc monosaccharide of the terminal unit [Gal-HexNAc] is linked to her in a way α1-3 or α 1-4 glycosidic

-

at another deoxyhexose radical, as long as it is present, the unit of HexNAc monosaccharide of the terminal unit [Gal-HexNAc] is a rhamnose radical and fucose

4. Use according to claim 1, characterized in that as an oligosaccharide lacto-N-tetraose is used (Galβ1-3GlcNAcβ1-3Galβ1-4Glc, LNT). 5. Use according to claim 4, characterized in that the lacto-N-tetraose is derivatized with fucose at an α-1-2 glycosidic junction in the terminal unit of monosaccharide galactose, and is lacto-N-fucopentaose I (LNFP I ). 6. Use according to the preceding claims, characterized in that the oligosaccharides are immobilized or adsorbed on an inert support. 7. Use according to claim 6, characterized in that the support is a peptide, a protein, a lipid, a polymer or a biopolymer. 8. Use according to one of the preceding claims, characterized in that the oligosaccharides are incorporated in a liquid or solid food (with the exception of human milk), dietary medium or pharmaceutical medium for administration in a person or an animal, or because it serves for the production of one such means. 9. Use according to claim 8, characterized in that the food is from a baby food or infant formula. 10. Use according to claim 8, characterized in that the pharmaceutical medium serves for oral, lingual, nasal, bronchial, vaginal, topical (skin and mucosa) or for administration by mouth or for administration by means of a probe in the stomach of a person or an animal. 11. Use according to one of the preceding claims, characterized in that the oligosaccharides are administered in an amount of at least 1 mg per kg of body weight of a person or an animal and day, but not in the form of human milk. 12. Use of a pharmaceutical medium according to one of the preceding claims, characterized in that the pharmaceutical media, apart from an auxiliary material or several auxiliary materials, including thickeners, humectants, diluents, flavoring materials, sweetening media and supports , so it can also contain another active principle or other active principles. 13. Use according to one of the preceding claims, characterized in that the food, dietary medium, or pharmaceutical medium is administered to a person or animal, although not in the form of human milk, in an amount such that it is supplied at least 1 mg of oligosaccharides according to one of claims 1 to 7, per day and per kilogram of body weight of the animal or person.